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College Park, Maryland      June 6 - 10 , 2004

WP7: A SANS Contrast Variation Study to Elucidate Structural Requirements for the Initial Recognition Step between Myosin Light Chain Kinase and Its Activatory Protein, Calmodulin

J.K. Krueger, N. Modi (University North Carolina Charlotte), W.T. Heller (Oak Ridge National Laboratory), D.J. Black, A. Persechini (University of Missouri Kansas City), J.T. Stull (University of Texas Southwestern Medical Center at Dallas), C.-S. Tung, J. Trewhella (Los Alamos National Laboratory)

A wide variety of Ca2+-dependent processes in the cell are regulated by the ubiquitous protein calmodulin via interactions with and subsequent activation of a diverse array of target proteins including a number of kinases, (proteins responsible for phosphorylation of other proteins). The Ca2+ -calmodulin (CaM)-dependent activation of myosin light chain kinase (MLCK) has served as a model for CaM-kinase interactions. We have collected small-angle neutron scattering data at several contrasts (0%, 15%, 30%, 70% and 80% D2O) on MLCK complexed with 60%-deuterated mutant CaM (delNCam). This mutant CaM missing an N-terminal leader sequence [residues #1-8 (A1DQLTEEQ8)] binds MLCK with high affinity but fails to activate catalysis. The small-angle scattering data reveal that delNCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Interestingly, the SANS data show that the CaM is in the collapsed state, suggesting that the delNCaM has bound to the autoinhibitory sequence of MLCK, rather than simply docking onto the surface of the MLCK in the extended state. A molecular model built from our SANS data will be used to provide the molecular boundaries for the individual protein components within the complex for which high-resolution crystal structures are known. The result will be a model of what is the initial recognition step in the CaM/MLCK interaction providing a detailed atomic description of the binding events between CaM and MLCK prior to the kinase activation step.

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